Compounds

ABSTRACT

The invention provides ERA polypeptides and DNA (RNA) encoding ERA polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing ERA polypeptide for the protection against infection, particularly bacterial infections.

RELATED APPLICATIONS

This application is a division of application Ser. No. 08/879,098, filedJun. 19,1997, now U.S. Pat. No. 6,248,556, which claims benefit of U.S.Provisional Application No. 60/011,888, filed Feb. 20, 1996, PCTApplication, International Application No. PCT/US97/02547, filed Feb.19, 1997, and PCT Application, International Application No.PCT/US97/02318, filed Feb. 19, 1997.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the GTP-binding protein family, hereinafter referred toas “ERA”.

BACKGROUND OF THE INVENTION

It is particularly preferred to employ Staphylococcal genes and geneproducts as targets for the development of antibiotics. TheStaphylococci make up a medically important genera of microbes. They areknown to produce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. S. aureus is the second leadingcause of bacteremia in cancer patients. Osteomyelitis, septic arthritis,septic thrombophlebitis and acute bacterial endocarditis are alsorelatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Staphylococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Staphylococcal food poisoning, scalded skin syndrome and toxic shocksyndrome.

A Blast P search result of publicly available sequence data, using theamino acid sequence of the invention as a query sequence, revealed aStreptococcus mutans homologue, which is a GTP-binding protein (ERA,sp/P42182/BEX_BACSU BEX PROTEIN).

Clearly, there is a need for factors that may be used to screencompounds for antibiotic activity, such as novel ERA of the invention.These factors may also be used to determine their roles in pathogenesisof infection, dysfunction and disease. There is a further need foridentification and characterization of such factors and theirantagonists and agonists which can play a role in preventing,ameliorating or correcting infections, dysfunctions or diseases.

The polypeptides of the invention have amino acid sequence homology to aknown Streptococcus mutans GTP-binding protein ERA protein.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel ERA polypeptides by homology between the amino acidsequence set out in FIG. 2 and a known amino acid sequence or sequencesof other proteins such as Streptococcus mutans GTP-binding protein ERAprotein.

It is a further object of the invention to provide polynucleotides thatencode ERA polypeptides, particularly polynucleotides that encode thepolypeptide herein designated ERA.

In a particularly preferred embodiment of this aspect of the inventionthe polynucleotide comprises a region encoding ERA polypeptidescomprising the sequence set out in FIG. 1 [SEQ ID NO: 1], or a variantthereof.

In another particularly preferred embodiment of the invention there is anovel ERA protein from Staphylococcus aureus comprising the amino acidsequence of FIG. 2 [SEQ ID NO: 2], or a variant thereof.

In accordance with this aspect of the invention there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the Staphylococcus aureus WCUH 29 strain contained in NCIMB DepositNo. 40771.

In accordance with this aspect of the invention there are providedisolated nucleic acid molecules encoding ERA, particularlyStaphylococcus aureus ERA, including mRNAs, cDNAs, genomic DNAs. Furtherembodiments of this aspect of the invention include biologically,diagnostically, prophylactically, clinically or therapeutically usefulvariants thereof, and compositions comprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of this aspect of the invention arenaturally occurring allelic variants of ERA and polypeptides encodedthereby.

In accordance with this aspect of the invention there are provided novelpolypeptides of Staphylococcus aureus referred to herein as ERA as wellas biologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of this aspect of theinvention are variants of ERA polypeptide encoded by naturally occurringalleles of the ERA gene.

In a preferred embodiment of this aspect of the invention there areprovided methods for producing the aforementioned ERA polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of this aspect of theinvention, there are provided products, compositions and methods for (i)assessing ERA expression, (ii) treating disease, for example, disease,such as, infections of the upper respiratory tract (e.g., otitis media,bacterial tracheitis, acute epiglottitis, thyroiditis), lowerrespiratory (e.g., empyema, lung abscess), cardiac (e.g., infectiveendocarditis), gastrointestinal (e.g., secretory diarrhoea, splenicabsces, retroperitoneal abscess), CNS (e.g., cerebral abscess), eye(e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis,preseptal and orbital cellulitis, darcryocystitis), kidney and urinarytract (e.g., epididymitis, intrarenal and perinephric absces, toxicshock syndrome), skin (e.g., impetigo, folliculitis, cutaneousabscesses, cellulitis, wound infection, bacterial myositis) bone andjoint (e.g., septic arthritis, osteomyelitis), (iii) assaying geneticvariation, (iv) and administering a ERA polypeptide or polynucleotide toan organism to raise an immunological response against a bacteria,especially a Staphylococcus aureus bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to ERA polynucleotide sequences, particularly under stringentconditions.

In certain preferred embodiments of this aspect of the invention thereare provided antibodies against ERA polypeptides.

In accordance with another aspect of the invention, there are providedERA agonists and antagonists each of which are preferably bacteriostaticor bacteriocidal.

In a further aspect of the invention there are provided compositionscomprising a ERA polynucleotide or a ERA polypeptide for administrationto a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings depict certain embodiments of the invention. Theyare illustrative only and do not limit the invention otherwise disclosedherein.

FIG. 1 shows a polynucleotide sequence of Staphylococcus aureus ERA [SEQID NO: 1].

FIG. 2 shows the amino acid sequence of Staphylococcus aureus ERA [SEQID NO: 2] deduced from the polynucleotide sequence of FIG. 1.

FIG. 3 shows a polynucleotide sequence embodiment of Staphylococcusaureus ERA [SEQ ID NO: 3].

FIG. 4 shows the amino acid sequence of Staphylococcus aureus ERA [SEQID NO: 4] deduced from the polynucleotide sequence of FIG. 3.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“Host cell” is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991). While there exist a number of methodsto measure identity and similarity between two sequences, both terms arewell known to skilled artisans (Sequence Analysis in Molecular Biology,von Heinje, G., Academic Press, 1987; Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988).Methods commonly employed to determine identity or similarity betweensequences include, but are not limited to those disclosed in Carillo,H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferredmethods to determine identity are designed to give the largest matchbetween the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include, but are not limited to, GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990)). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990)).

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded, or a mixture of single- and double-stranded regions. Inaddition, polynucleotide as used herein refers to triple-strandedregions comprising RNA or DNA or both RNA and DNA. The strands in suchregions may be from the same molecule or from different molecules. Theregions may include all of one or more of the molecules, but moretypically involve only a region of some of the molecules. One of themolecules of a triple-helical region often is an oligonucleotide. Asused herein, the term “polynucleotide(s)” includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” embraces short polynucleotides often referred to asoligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques which are well known to the art. Such modifications are welldescribed in basic texts and in more detailed monographs, as well as ina voluminous research literature, and they are well known to those ofskill in the art. It will be appreciated that the same type ofmodification may be present in the same or varying degree at severalsites in a given polypeptide. Also, a given polypeptide may contain manytypes of modifications. Modifications can occur anywhere in apolypeptide, including the peptide backbone, the amino acid side-chainsand the amino or carboxyl termini. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation andADP-ribosylation, selenoylation, sulfation, transfer-RNA mediatedaddition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork (1993) and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York(1983); Seifter et al., Meth. Enzymol. 182: 626-646 (1990) and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched orcyclic, with or without branching. Cyclic, branched and branchedcircular polypeptides may result from posttranslational naturalprocesses and may be made by entirely synthetic methods, as well.

“Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF THE INVENTION

The invention relates to novel ERA polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a novel ERA gene ofStaphylococcus aureus, which is related by amino acid sequence homologyto Streptococcus mutans GTP-binding protein ERA polypeptide. Theinvention relates especially to ERA having the nucleotide and amino acidsequences set out in FIG. 1 and FIG. 2 respectively, and to the ERAnucleotide sequences of the DNA in NCIMB Deposit No. 40771 and aminoacid sequences encoded thereby.

Techniques are available to evaluate temporal gene expression inbacteria, particularly as it applies to viability under laboratory andhost infection conditions. A number of methods can be used to identifygenes which are essential to survival per se, or essential to theestablishment and/or maintenance of an infection. Identification ofexpression of a sequence by one of these methods yields additionalinformation about its function and assists in the selection of suchsequence for further development as a screening target. Briefly, theseapproaches include for example;

1) Signature Tagged Mutagenesis (STM)

This technique is described by Hensel et al., Science 269:400-403(1995), the contents of which is incorporated by reference forbackground purposes. Signature tagged mutagenesis identifies genesnecessary for the establishment/maintenance of infection in a giveninfection model.

The basis of the technique is the random mutagenesis of target organismby various means (e.g., transposons) such that unique DNA sequence tagsare inserted in close proximity to the site of mutation. The tags from amixed population of bacterial mutants and bacteria recovered from aninfected hosts are detected by amplification, radiolabeling andhybridization analysis. Mutants attenuated in virulence are revealed byabsence of the tag from the pool of bacteria recovered from infectedhosts.

In Staphylococcus aureus, because the transposon system is less welldeveloped, a more efficient way of creating the tagged mutants is to usethe insertion-duplication mutagenesis technique as described in Morrisonet al., J. Bacteriol. 159: 870 (1984) the contents of which isincorporated by reference for background purposes.

2) In Vivo Expression Technology (IVET)

This technique is described by Camilli et al., Proc. Nat'l. Acad. Sci.USA. 91: 2634-2638 (1994) and Mahan et al., Infectious Agents andDiseases 2: 263-268 (1994), the contents of each of which isincorporated by reference for background purposes. IVET identifies genesup-regulated during infection when compared to laboratory cultivation,implying an important role in infection. Sequences identified by thistechnique are implied to have a significant role in infectionestablishment/maintenance.

In this technique random chromosomal fragments of target organism arecloned upstream of a promoter-less reporter gene in a plasmid vector.The pool is introduced into a host and at various times after infectionbacteria may be recovered and assessed for the presence of reporter geneexpression. The chromosomal fragment carried upstream of an expressedreporter gene should carry a promoter or portion of a gene normallyupregulated during infection. Sequencing upstream of the reporter geneallows identification of the up regulated gene.

3) Differential display

This technique is described in Chuang et al., J. Bacteriol. 175:2026-2036 (1993), the contents of which is incorporated by reference forbackground purposes. This method identifies those genes which areexpressed in an organism by identifying mRNA present usingrandomly-primed RT-PCR. By comparing pre-infection and post infectionprofiles, genes up and down regulated during infection can be identifiedand the RT-PCR product sequenced and matched to library sequences.

4) Generation of conditional lethal mutants by transposon mutagenesis.

This technique, described by de Lorenzo, V. et al., Gene 123: 17-24(1993); Neuwald, A. F. et al., Gene 125: 69-73(1993); and Takiff, H. E.et al., J. Bacteriol. 174: 1544-1553(1992), the contents of which isincorporated by reference for background purposes, identifies geneswhose expression are essential for cell viability.

In this technique transposons carrying controllable promoters, whichprovide transcription outward from the transposon in one or bothdirections, are generated. Random insertion of these transposons intotarget organisms and subsequent isolation of insertion mutants in thepresence of inducer of promoter activity ensures that insertions whichseparate promoter from coding region of a gene whose expression isessential for cell viability will be recovered. Subsequent replicaplating in the absence of inducer identifies such insertions, since theyfail to survive. Sequencing of the flanking regions of the transposonallows identification of site of insertion and identification of thegene disrupted. Close monitoring of the changes in cellularprocesses/morphology during growth in the absence of inducer yieldsinformation on likely function of the gene. Such monitoring couldinclude flow cytometry (cell division, lysis, redox potential, DNAreplication), incorporation of radiochemically labeled precursors intoDNA, RNA, protein, lipid, peptidoglycan, monitoring reporter enzyme genefusions which respond to known cellular stresses.

5) Generation of conditional lethal mutants by chemical mutagenesis.

This technique is described by Beckwith , J., Methods in Enzymology 204:3-18(1991), the contents of which are incorporated herein by referencefor background purposes. In this technique random chemical mutagenesisof target organism, growth at temperature other than physiologicaltemperature (permissive temperature) and subsequent replica plating andgrowth at different temperature (e.g., 42° C. to identify ts, 25° C. toidentify cs) are used to identify those isolates which now fail to grow(conditional mutants). As above close monitoring of the changes upongrowth at the non-permissive temperature yields information on thefunction of the mutated gene. Complementation of conditional lethalmutation by library from target organism and sequencing of complementinggene allows matching with library sequences.

6) RT-PCR

Bacterial messenger RNA, preferably that of Staphylococcus aureus, isisolated from bacterial infected tissue, e.g., 48 hour murine lunginfections, and the amount of each mRNA species assessed by reversetranscription of the RNA sample primed with random hexanucleotidesfollowed by PCR with gene specific primer pairs. The determination ofthe presence and amount of a particular mRNA species by quantificationof the resultant PCR product provides information on the bacterial geneswhich are transcribed in the infected tissue. Analysis of genetranscription can be carried out at different times of infection to gaina detailed knowledge of gene regulation in bacterial pathogenesisallowing for a clearer understanding of which gene products representtargets for screens for novel antibacterials. Because of the genespecific nature of the PCR primers employed it should be understood thatthe bacterial mRNA preparation need not be free of mammalian RNA. Thisallows the investigator to carry out a simple and quick RNA preparationfrom infected tissue to obtain bacterial mRNA species which are veryshort lived in the bacterium (in the order of 2 minute halflives).Optimally the bacterial mRNA is prepared from infected murine lungtissue by mechanical disruption in the presence of TRIzole (GIBCO-BRL)for very short periods of time, subsequent processing according to themanufacturers of TRIzole reagent and DNAase treatment to removecontaminating DNA. Preferably the process is optimized by finding thoseconditions which give a maximum amount of bacterial 16 S ribosomal RNA,preferably that of Staphylococcus aureus, as detected by probingNortherns with a suitably labeled sequence specific oligonucleotideprobe. Typically, a 5′ dye labelled primer is used in each PCR primerpair in a PCR reaction which is terminated optimally between 8 and 25cycles. The PCR products are separated on 6% polyacrylamide gels withdetection and quantification using GeneScanner (manufactured by ABI).

Each of these techniques may have advantages or disadvantage dependingon the particular application. The skilled artisan would choose theapproach that is the most relevant with the particular end use in mind.For example, some genes might be recognised as essential for infectionbut in reality are only necessary for the initiation of infection and sotheir products would represent relatively unattractive targets forantibacterials developed to cure established and chronic infections.

Use of the of these technologies when applied to the sequences of theinvention enables identification of bacterial proteins expressed duringinfection, inhibitors of which would have utility in anti-bacterialtherapy.

Deposited Materials

A deposit containing a Staphylococcus aureus WCUH 29 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (NCIMB), 23 St. Machar Drive, Aberdeen AB2 1RY, Scotlandon Sep. 11, 1995 and assigned NCIMB Deposit No. 40771. TheStaphylococcus aureus strain deposit is referred to herein as “thedeposited strain” or as “the DNA of the deposited strain”.

The deposited material is a strain that contains the full length ERADNA, referred to as “NCIMB 40771” upon deposit.

The sequence of the polynucleotides contained in the deposited material,as well as the amino acid sequence of the polypeptide encoded thereby,are controlling in the event of any conflict with any description ofsequences herein.

The deposit has been made under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Micro-organisms for Purposesof Patent Procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and is not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. §112.

A license may be required to make, use or sell the deposited materials,and no such license is hereby granted.

Polypeptides

The polypeptides of the invention include the polypeptide of FIG. 2 [SEQID NO: 2] (in particular the mature polypeptide) as well as polypeptidesand fragments, such as the polypeptide of SEQ ID NO: 4, and particularlythose polypeptides which have the biological activity of ERA, and alsothose which have at least 70% identity to the polypeptide of FIG. 2 [SEQID NO: 2] or FIG. 4 [SEQ ID NO: 4] or the relevant portion, preferablyat least 80% identity to the polypeptide of FIG. 2 [SEQ ID NO: 2] orFIG. 4 [SEQ ID NO: 4], and more preferably at least 90% similarity (morepreferably at least 90% identity) to the polypeptide of FIG. 2 [SEQ IDNO: 2] or FIG. 4 [SEQ ID NO: 4] and still more preferably at least 95%similarity (still more preferably at least 95% identity) to thepolypeptide of FIG. 2 [SEQ ID NO: 2] or FIG. 4 [SEQ ID NO: 4] and alsoinclude portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

Variants that are fragments of the polypeptides of the invention may beemployed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides. Variants thatare fragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

A fragment is a variant polypeptide having an amino acid sequence thatentirely is the same as part but not all of the amino acid sequence ofthe aforementioned polypeptides. As with ERA polypeptides fragments maybe “free-standing”, or comprised within a larger polypeptide of whichthey form a part or region, most preferably as a single continuousregion, a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of the amino acid sequence of FIG. 2 [SEQ ID NO: 2], or ofvariants thereof, except for deletion of a continuous series of residuesthat includes the amino terminus, or a continuous series of residuesthat includes the carboxyl terminus or deletion of two continuous seriesof residues, one including the amino terminus and one including thecarboxyl terminus. Degradation forms of the polypeptides of theinvention in a host cell, particularly a Staphylococcus aureus, are alsopreferred. Also preferred are fragments characterized by structural orfunctional attributes such as fragments that comprise alpha-helix andalpha-helix forming regions, beta-sheet and beta-sheet-forming regions,turn and turn-forming regions, coil and coil-forming regions,hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions.

Also preferred are biologically active fragments which are thosefragments that mediate activities of ERA, including those with a similaractivity or an improved activity, or with a decreased undesirableactivity. Also included are those fragments that are antigenic orimmunogenic in an animal, especially in a human.

Polynucleotides

Another aspect of the invention relates to isolated polynucleotideswhich encode the ERA polypeptide having the deduced amino acid sequenceof FIG. 2 [SEQ ID NO: 2] and polynucleotides closely related thereto andvariants thereto.

Using the information provided herein, such as the polynucleotidesequence set out in FIG. 1 [SEQ ID NO: 1], a polynucleotide of theinvention encoding ERA polypeptide may be obtained using standardcloning and screening, such as those for cloning and sequencingchromosomal DNA fragments from Staphylococcus aureus WCUH 29 cells asstarting material, followed by obtaining a full length clone. Forexample, to obtain a polynucleotide sequence of the invention, such asthat sequence given in FIG. 1 [SEQ ID NO: 1]or FIG. 3 [SEQ ID NO: 3],typically a library of clones of chromosomal DNA of Staphylococcusaureus WCUH 29 in E. coli or some other suitable host is probed with aradiolabeled oligonucleotide, preferably a 17-mer or longer, derivedfrom a partial sequence. Clones carrying DNA identical to that of theprobe can then be distinguished using stringent conditions. Bysequencing the individual clones thus identified with sequencing primersdesigned from the original sequence it is then possible to extend thesequence in both directions to determine the full gene sequence.Conveniently such sequencing is performed using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see Screening By Hybridization1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70).Illustrative of the invention, the polynucleotide set out in FIG. 1 [SEQID NO: 1] was discovered in a DNA library derived from Staphylococcusaureus WCUH 29.

The DNA sequence thus obtained is set out in FIG. 1 [SEQ ID NO: 1]. Itcontains an open reading frame encoding a protein having about thenumber of amino acid residues set forth in FIG. 2 [SEQ ID NO: 2] with adeduced molecular weight that can be calculated using amino acid residuemolecular weight values well known in the art.

ERA of the invention is structurally related to other proteins of theGTP-binding protein family, as shown by the results of sequencing theDNA encoding ERA of the deposited strain. The protein exhibits greatesthomology to Streptococcus mutans GTP-binding protein ERA protein amongknown proteins. ERA of FIG. 2 [SEQ ID NO: 2] has about 61% identity overits entire length and about 79% similarity over its entire length withthe amino acid sequence of Streptococcus mutans GTP-binding protein ERA.

Sequence of the invention may also be identical over its entire lengthto the coding sequence in FIG. 1 [SEQ ID NO: 1] or FIG. 3 [SEQ ID NO:3]. The sequence of FIG. 3 [SEQ ID NO: 3], from nucleotides 242 to 523,putatively encodes the polypeptide of FIG. 4 [SEQ ID NO: 4].

Also provided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro- protein sequence. Thepolynucleotide may also contain non-coding sequences, including forexample, but not limited to non-coding 5′ and 3′ sequences, such as thetranscribed, non-translated sequences, termination signals, ribosomebinding sites, sequences that stabilize mRNA, introns, polyadenylationsignals, and additional coding sequence which encode additional aminoacids. For example, a marker sequence that facilitates purification ofthe fused polypeptide can be encoded. In certain embodiments of thisaspect of the invention, the marker sequence is a hexa-histidinepeptide, as provided in the pQE vector (Qiagen, Inc.) and described inGentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824 (1989), or an HAtag (Wilson et al., Cell 37: 767 (1984)). Polynucleotides of theinvention also include, but are not limited to, polynucleotidescomprising a structural gene and its naturally associated sequences thatcontrol gene expression.

In accordance with the foregoing, the term “polynucleotide encoding apolypeptide” as used herein encompasses polynucleotides which include asequence encoding a polypeptide of the invention, particularlybacterial, and more particularly the Staphylococcus aureus ERA havingthe amino acid sequence set out in FIG. 2 [SEQ ID NO: 2]. The termencompasses polynucleotides that include a single continuous region ordiscontinuous regions encoding the polypeptide (for example, interruptedby integrated phage or an insertion sequence or editing) together withadditional regions, that also may contain coding and/or non-codingsequences.

The invention further relates to variants of the herein above describedpolynucleotides which encode for variants of the polypeptide having thededuced amino acid sequence of FIG. 2 [SEQ ID NO:2].

Further particularly preferred embodiments are polynucleotides encodingERA variants, which have the amino acid sequence of ERA polypeptide ofFIG. 2 [SEQ ID NO: 2] in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, which do not alter the properties andactivities of ERA.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding ERA polypeptide having the amino acid sequence set out in FIG.2 [SEQ ID NO: 2], and polynucleotides which are complementary to suchpolynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover their entire length to a polynucleotide encoding ERA polypeptide ofthe Staphylococcus aureus DNA of the deposited strain andpolynucleotides complementary thereto. In this regard, polynucleotidesat least 90% identical over their entire length to the same areparticularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

Preferred embodiments in this respect, moreover, are polynucleotideswhich encode polypeptides which retain substantially the same biologicalfunction or activity as the mature polypeptide encoded by the DNA ofFIG. 1 [SEQ ID NO: 1].

The invention further relates to polynucleotides that hybridize to theherein above-described sequences. In this regard, the inventionespecially relates to polynucleotides which hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms “stringent conditions” and “stringent hybridizationconditions” mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the filters in 0.1× SSC at about65° C. Hybridization and wash conditions are well known and exemplifiedin Sambrook, et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11therein, the disclosure of which is hereby incorporated in its entiretyby reference.

The invention also provides a polynucleotide consisting essentially of apolynucleotide sequence obtainable by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO: 1 under stringent hybridization conditions with a probehaving the sequence of said polynucleotide sequence set forth in SEQ IDNO: 1 or a fragment thereof, and isolating said DNA sequence. Fragmentsuseful for obtaining such a polynucleotide include, for example, probesand primers described elsewhere herein.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding ERA and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the ERA gene. Such probes generally will comprise at least15 bases. Preferably, such probes will have at least 30 bases and mayhave at least 50 bases. Particularly preferred probes will have at least30 bases and will have 50 bases or less.

For example, the coding region of the ERA gene may be isolated byscreening using the known DNA sequence to synthesize an oligonucleotideprobe. A labeled oligonucleotide having a sequence complementary to thatof a gene of the invention is then used to screen a library of cDNA,genomic DNA or mRNA to determine which members of the library the probehybridizes to.

The polynucleotides and polypeptides of the invention may be employed asresearch reagents and materials for discovery of treatments of anddiagnostics for disease, particularly human disease, as furtherdiscussed herein relating to polynucleotide assays, inter alia.

Polynucleotides of the invention that are oligonucleotides derived fromthe sequences of SEQ ID NOS: 1 and 2 may be used in the processes hereinas described, but preferably for PCR, to determine whether or not thepolynucleotides identified herein in whole or in part are transcribed ininfected tissue. It is recognized that such sequences will also haveutility in diagnosis of the stage of infection and type of infection thepathogen has attained.

The polynucleotides may encode a polypeptide which is the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature polypeptide (when the mature form has more thanone polypeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, may allowprotein transport, may lengthen or shorten protein half-life or mayfacilitate manipulation of a protein for assay or production, amongother things. As generally is the case in vivo, the additional aminoacids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences which are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, host cells, expression

The invention also relates to vectors which comprise a polynucleotide orpolynucleotides of the invention, host cells which are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation system can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof or polynucleotides ofthe invention. Introduction of a polynucleotide into the host cell canbe effected by methods described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), suchas, calcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used to produce apolypeptide of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic Assays

This invention is also related to the use of the ERA polynucleotides ofthe invention for use as diagnostic reagents. Detection of ERA in aeukaryote, particularly a mammal, and especially a human, will provide adiagnostic method for diagnosis of a disease. Eukaryotes (herein also“individual(s)”), particularly mammals, and especially humans, infectedwith an organism comprising the ERA gene may be detected at the DNAlevel by a variety of techniques.

Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the strain of prokaryote present in aeukaryote, particularly a mammal, and especially a human, may be made byan analysis of the genotype of the prokaryote gene. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the genotype of a reference sequence. Point mutationscan be identified by hybridizing amplified DNA to labeled ERApolynucleotide sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase digestion or bydifferences in melting temperatures. DNA sequence differences may alsobe detected by alterations in the electrophoretic mobility of the DNAfragments in gels, with or without denaturing agents, or by direct DNAsequencing. See, e.g., Myers et al., Science, 230: 1242 (1985). Sequencechanges at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or a chemicalcleavage method. See, e.g., Cotton et al., Proc. Natl. Acad. Sci., USA,85: 4397-4401 (1985).

Cells carrying mutations or polymorphisms in the gene of the inventionmay also be detected at the DNA level by a variety of techniques, toallow for serotyping, for example. For example, RT-PCR can be used todetect mutations. It is particularly preferred to used RT-PCR inconjunction with automated detection systems, such as, for example,GeneScan. RNA or cDNA may also be used for the same purpose, PCR orRT-PCR. As an example, PCR primers complementary to the nucleic acidencoding ERA can be used to identify and analyze mutations.

These primers may also be used for amplifying ERA DNA isolated from asample derived from an individual. The invention further provides theseprimers with 1, 2, 3 or 4 nucleotides removed from the 5′ and/or the 3′end. The primers may be used to amplify the gene isolated from aninfected individual such that the gene may then be subject to varioustechniques for elucidation of the DNA sequence. In this way, mutationsin the DNA sequence may be detected and used to diagnose infection andto serotype or classify the infectious agent.

The invention provides a process for diagnosing, disease, preferablybacterial infections, more preferably infections by Staphylococcusaureus, and most preferably disease, such as, infections of the upperrespiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric absces, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis), comprising determining from a sample derived from anindividual a increased level of expression of polynucleotide having thesequence of FIG. 1 [SEQ ID NO: 1]. Increased or decreased expression ofERA polynucleotide can be measured using any on of the methods wellknown in the art for the quantation of polynucleotides, such as, forexample, amplification, PCR, RT-PCR, RNase protection, Northern blottingand other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of ERA protein compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a ERAprotein, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. “Antibodies” as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art which provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology could be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-Fbp or from naive libraries (McCafferty, J.et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope—termed ‘bispecific’ antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against ERA may be employed to treatinfections, particularly bacterial infections and especially disease,such as, infections of the upper respiratory tract (e.g., otitis media,bacterial tracheitis, acute epiglottitis, thyroiditis), lowerrespiratory (e.g., empyema, lung abscess), cardiac (e.g., infectiveendocarditis), gastrointestinal (e.g., secretory diarrhoea, splenicabsces, retroperitoneal abscess), CNS (e.g., cerebral abscess), eye(e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis,preseptal and orbital cellulitis, darcryocystitis), kidney and urinarytract (e.g., epididymitis, intrarenal and perinephric absces, toxicshock syndrome), skin (e.g., impetigo, folliculitis, cutaneousabscesses, cellulitis, wound infection, bacterial myositis) bone andjoint (e.g., septic arthritis, osteomyelitis).

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants which form a particular aspect ofthis invention. The term “antigenically equivalent derivative” as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognised by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm “immunologically equivalent derivative” as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be “humanized”; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1: 363, Manthorpe et al., Hum. Gene Ther. 1963: 4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem 1989:264, 16985), coprecipitation of DNA with calcium phosphate (Benvenisty &Reshef, PNAS, 1986: 83, 9551), encapsulation of DNA in various forms ofliposomes (Kaneda et al., Science 1989: 243, 375), particle bombardment(Tang et al., Nature 1992, 356: 152, Eisenbraun et al., DNA Cell Biol1993, 12: 791) and in vivo infection using cloned retroviral vectors(Seeger et al., PNAS 1984: 81,5849).

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

Antagonists and agonists—assays and molecules

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action of ERApolypeptides or polynucleotides.

For example, to screen for agonists or antagoists, a synthetic reactionmix, a cellular compartment, such as a membrane, cell envelope or cellwall, or a preparation of any thereof, comprising ERA polypeptide and alabeled substrate or ligand of such polypeptide is incubated in theabsence or the presence of a candidate molecule which may be a ERAagonist or antagonist. The ability of the candidate molecule to agonizeor antagonize the ERA polypeptide is reflected in decreased binding ofthe labeled ligand or decreased production of product from suchsubstrate. Molecules which bind gratuitously, i.e., without inducing theeffects of ERA are most likely to be good antagonists. Molecules thatbind well and increase the rate of product production from substrate areagonists. The rate or level or production of product from substrate maybe enhanced by using a reporter system. Reporter systems that may beuseful in this regard include but are not limited to colorimetriclabeled substrate converted into product, a reporter gene that isresponsive to changes in ERA activity, and binding assays known in theart.

Another example of an assay for ERA antagonists is a competitive assaythat combines ERA and a potential antagonist with ERA-binding molecules,recombinant ERA binding molecules, natural substrate or ligands, orsubstrate or ligand mimetics, under appropriate conditions for acompetitive inhibition assay. ERA can be labeled, such as byradioactivity or a colorimetric compound, such that the number of ERAmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a binding molecule, without inducingERA-induced activities, thereby preventing the action of ERA byexcluding ERA from binding.

Potential antagonists include a small molecule which binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of ERA.

In a particular aspect the invention provides the use of thepolypeptide, polynucleotide or inhibitor of the invention to interferewith the initial physical interaction between a pathogen and mammalianhost responsible for sequelae of infection. In particular the moleculesof the invention may be used: (i) in the prevention of adhesion ofbacteria, in particular gram positive bacteria, to mammalianextracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; (ii) to block ERA protein mediated mammaliancell invasion by, for example, initiating phosphorylation of mammaliantyrosine kinases (Rosenshine et al., Infect. Immun. 60: 2211 (1992);(iii) to block bacterial adhesion between mammalian extracellular matrixproteins and bacterial ERA proteins which mediate tissue damage; and(iv) to block the normal progression of pathogenesis in infectionsinitiated other than by the implantation of in-dwelling devices or byother surgical techniques.

Each of the DNA sequences provided herein may be used in discovery anddevelopment of antibacterial compounds. The encoded protein uponexpression can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequences ofinterest.

The antagonists and agonists may be employed for instance to inhibitdisease, such as, infections of the upper respiratory tract (e.g.,otitis media, bacterial tracheitis, acute epiglottitis, thyroiditis),lower respiratory (e.g., empyema, lung abscess), cardiac (e.g.,infective endocarditis), gastrointestinal (e.g., secretory diarrhoea,splenic abscess, retroperitoneal absces), CNS (e.g., cerebral abscess),eye (e.g., blepharitis, conjunctivitis, keratitis, endophthalmitis,preseptal and orbital cellulitis, darcryocystitis), kidney and urinarytract (e.g., epididymitis, intrarenal and perinephric absces, toxicshock syndrome), skin (e.g., impetigo, folliculitis, cutaneousabscesses, cellulitis, wound infection, bacterial myositis) bone andjoint (e.g., septic arthritis, osteomyelitis).

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with ERA, or a fragment or variantthereof, adequate to produce antibody to protect said individual frominfection, particularly bacterial infection and most particularlyStaphylococcus aureus infections. Yet another aspect of the inventionrelates to a method of inducing immunological response in an individualwhich comprises, through gene therapy, delivering gene encoding ERA, ora fragment or a variant thereof, for expressing ERA, or a fragment or avariant thereof in vivo in order to induce an immunological response toproduce antibody to protect said individual from disease.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into a host capable or having inducedwithin it an immunological response, induces an immunological responsein such host to a ERA or protein coded therefrom, wherein thecomposition comprises a recombinant ERA or protein coded therefromcomprising DNA which codes for and expresses an antigen of said ERA orprotein coded therefrom.

The ERA or a fragment thereof may be fused with co-protein which may notby itself produce antibodies, but is capable of stabilizing the firstprotein and producing a fused protein which will have immunogenic andprotective properties. Thus fused recombinant protein, preferablyfurther comprises an antigenic co-protein, such asGlutathione-S-transferase (GST) or beta-galactosidase, relatively largeco-proteins which solubilise the protein and facilitate production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system.The co-protein may be attached to either the amino or carboxy terminusof the first protein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al., Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Staphylococcus aureus will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believe that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStaphylococcus aureus infections, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises theimmunogenic recombinant protein together with a suitable carrier. Sincethe protein may be broken down in the stomach, it is preferablyadministered parenterally, including, for example, administration thatis subcutaneous, intramuscular, intravenous, or intradermal.Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation instonicwith the bodily fluid, preferably the blood, of the individual; andaqueous and non-aqueous sterile suspensions which may include suspendingagents or thickening agents. The formulations may be presented inunit-dose or multiple-dose containers, for example, sealed ampoules andvials and may be stored in a freeze-dried condition requiring only theaddition of the sterile liquid carrier immediately prior to use. Thevaccine formulation may also include adjuvant systems for enhancing theimmunogenicity of the formulation, such as oil-in water systems andother system known in the art. The dosage will depend on the specificactivity of the vaccine and can be readily determined by routineexperimentation.

While the invention has been described with reference to certain ERA, itis to be understood that this covers fragments of the naturallyoccurring protein and similar proteins with additions, deletions orsubstitutions which do not substantially affect the immunogenicproperties of the recombinant protein.

Compositions, kits and administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or the agonists or antagonists. Thepolypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject. Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters,etc.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStaphylococcus aureus wound infections.

Many orthopedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 μg/kg of antigen, and such dose ispreferably administered 1-3 times and with an interval of 1-3 weeks.With the indicated dose range, no adverse toxicological effects will beobserved with the compounds of the invention which would preclude theiradministration to suitable individuals.

The antibodies described above may also be used as diagnostic reagentsto detect the presence of bacteria containing the ERA protein.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Library Production

The polynucleotide having the DNA sequence given in SEQ ID NO: 1 wasobtained from a library of clones of chromosomal DNA of Staphylococcusaureus in E. coli. In some cases the sequencing data from two or moreclones containing overlapping Staphylococcus aureus DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO: 1. Libraries may beprepared by routine methods using, for example, Methods 1 or 2 below:

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either offollowing two methods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackage library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagelibrary. The library is amplified by standard procedures.

4 900 base pairs nucleic acid double linear 1 ATGACAGAAC ATAAATCAGGATTTGTTTCA ATTATAGGTA GACCAAATGT AGGGAAGTCA 60 ACATTTGTTA ATAGAGTGATTGGCCATAAA ATAGCAATCA TGTCCGATAA AGCTCAAACA 120 ACTAGAAATA AAATTCAAGGTGTTATGACA AGAGATGACG CGCAAATTAT ATTCAATGAT 180 ACGCCAGGTA TTCATAAACCTAAACACAAA TTAGGTGACT ATATGATGAA AGTCGCTAAA 240 AATACATTAT CTGAAATAGATGCAATCATG TTTATGGTTA ATGCCAATGA GGAAATTGGA 300 CGAGGCGATG AATATATTATAGAAATGTTG AAAAATGTTA AGACACCAGT ATTTTTAGTA 360 TTAAATAAAA TAGATTTAGTGCATCCAGAT GAATTAATGC CAAAGATTGA AGAATATCAA 420 AGTTATATGG ACTTTACAGAGATTGTACCT ATTTCAGCAT TAGAAGGGCT AAATGTAGAT 480 CATTTTATTG ATGTTTTAAAGACGTATTTA CCCGAAGGAC CTAAATATTA TCCAGATGAT 540 CAAATTTCAG ACCATCCTGAACAATTTGTA GTGGGTGAAA TCATTCGTGA AAAAATCCTT 600 CATCTTACAA GTGAAGAAATCCCTCATGCG ATTGGTGTTA ATGTGGACCG TATGGTTAAA 660 GAAAGCGAAG ATCGTGTTCATATCGAAGCA ACTATATATG TTGAAAGAGA TTCGCAAAAA 720 GGAATTGTCA TTGGAAAAGGCGGTAAAAAG TTAAAAGAAG TAGGAAAACG TGCGAGACGT 780 GATATAGAAA TGCTTCTAGGCTCTAAAGTA TACTTAGAAT TATGGGTCAA AGTTCAAAGA 840 GACTGGCGAA ACAAAGTTAACTTTATTCGC CAAATTGGTT ATGTTGAAGA CCAAGATTAA 900 299 amino acids aminoacid single linear 2 Met Thr Glu His Lys Ser Gly Phe Val Ser Ile Ile GlyArg Pro Asn 1 5 10 15 Val Gly Lys Ser Thr Phe Val Asn Arg Val Ile GlyHis Lys Ile Ala 20 25 30 Ile Met Ser Asp Lys Ala Gln Thr Thr Arg Asn LysIle Gln Gly Val 35 40 45 Met Thr Arg Asp Asp Ala Gln Ile Ile Phe Asn AspThr Pro Gly Ile 50 55 60 His Lys Pro Lys His Lys Leu Gly Asp Tyr Met MetLys Val Ala Lys 65 70 75 80 Asn Thr Leu Ser Glu Ile Asp Ala Ile Met PheMet Val Asn Ala Asn 85 90 95 Glu Glu Ile Gly Arg Gly Asp Glu Tyr Ile IleGlu Met Leu Lys Asn 100 105 110 Val Lys Thr Pro Val Phe Leu Val Leu AsnLys Ile Asp Leu Val His 115 120 125 Pro Asp Glu Leu Met Pro Lys Ile GluGlu Tyr Gln Ser Tyr Met Asp 130 135 140 Phe Thr Glu Ile Val Pro Ile SerAla Leu Glu Gly Leu Asn Val Asp 145 150 155 160 His Phe Ile Asp Val LeuLys Thr Tyr Leu Pro Glu Gly Pro Lys Tyr 165 170 175 Tyr Pro Asp Asp GlnIle Ser Asp His Pro Glu Gln Phe Val Val Gly 180 185 190 Glu Ile Ile ArgGlu Lys Ile Leu His Leu Thr Ser Glu Glu Ile Pro 195 200 205 His Ala IleGly Val Asn Val Asp Arg Met Val Lys Glu Ser Glu Asp 210 215 220 Arg ValHis Ile Glu Ala Thr Ile Tyr Val Glu Arg Asp Ser Gln Lys 225 230 235 240Gly Ile Val Ile Gly Lys Gly Gly Lys Lys Leu Lys Glu Val Gly Lys 245 250255 Arg Ala Arg Arg Asp Ile Glu Met Leu Leu Gly Ser Lys Val Tyr Leu 260265 270 Glu Leu Trp Val Lys Val Gln Arg Asp Trp Arg Asn Lys Val Asn Phe275 280 285 Ile Arg Gln Ile Gly Tyr Val Glu Asp Gln Asp 290 295 540 basepairs nucleic acid double linear 3 GGCACGAGCT AGTTTGGTAT CGGCAATTTCTCAAGGATAC AGACCAGGTG ATTTTGAATC 60 AATAACTGTA ACCGTAGATG CAGATAAACCGTCATCACCT TGTGGTGCAT GTCGTCAAGT 120 TTTAAAGGAA TTATGTGATG ATGATATGCCTGTGTATATG ACAAATCATA AAGGAGATAT 180 GGTTATGATG ACAGNCGCAG AGTTACTACCATTTGGATTT TCAGGAAAGG ATTTAGAATA 240 AATGACAGAA CATAAATCAG GATTTGTTTCAATTATAGGT AGACCAAATG TAGGGAAGTC 300 AACATTTGTT AATAGAGTGA TTGGCCATAAAATAGCAATC ATGTCCGATA AAGCTCAAAC 360 AACTAGAAAT AAAATTCAAG GTGTTATGACAAGAGATGAC GCGCAAATTA TATTCAATGA 420 TACGCCAGGT ATTCATAAAC CTAAACACAAATTAGGTGAT TATACTGATG AAAGTCGCTT 480 AAAATACATA TCTGAAATAG ATGCAATCATGGTTTATGGC TAATTGCAAT GAGAAATTGG 540 93 amino acids amino acid singlelinear 4 Met Thr Glu His Lys Ser Gly Phe Val Ser Ile Ile Gly Arg Pro Asn1 5 10 15 Val Gly Lys Ser Thr Phe Val Asn Arg Val Ile Gly His Lys IleAla 20 25 30 Ile Met Ser Asp Lys Ala Gln Thr Thr Arg Asn Lys Ile Gln GlyVal 35 40 45 Met Thr Arg Asp Asp Ala Gln Ile Ile Phe Asn Asp Thr Pro GlyIle 50 55 60 His Lys Pro Lys His Lys Leu Gly Asp Tyr Thr Asp Glu Ser ArgLeu 65 70 75 80 Lys Tyr Ile Ser Glu Ile Asp Ala Ile Met Val Tyr Gly 8590

What is claimed is:
 1. An isolated polypeptide comprising SEQ ID NO: 2.2. A composition comprising the isolated polypeptide of claim 1 and acarrier.
 3. The isolated polypeptide of claim 1, wherein the isolatedpolypeptide comprises a heterologous amino acid sequence fused to SEQ IDNO:
 2. 4. A composition comprising the isolated polypeptide of claim 3and a carrier.
 5. The isolated polypeptide of claim 1, wherein theisolated polypeptide consists of SEQ ID NO:
 2. 6. A compositioncomprising the isolated polypeptide of claim 5 and a carrier.